The modular nature of histone deacetylase HDAC4 confers phosphorylation-dependent intracellular trafficking.

In C2C12 myoblasts, endogenous histone deacetylase HDAC4 shuttles between cytoplasmic and nuclear compartments, supporting the hypothesis that its subcellular localization is dynamically regulated. However, upon differentiation, this dynamic equilibrium is disturbed and we find that HDAC4 accumulates in the nuclei of myotubes, suggesting a positive role of nuclear HDAC4 in muscle differentiation. Consistent with the notion of regulation of HDAC4 intracellular trafficking, we reveal that HDAC4 contains a modular structure consisting of a C-terminal autonomous nuclear export domain, which, in conjunction with an internal regulatory domain responsive to calcium/calmodulin-dependent protein kinase IV (CaMKIV), determines its subcellular localization. CaMKIV phosphorylates HDAC4 in vitro and promotes its nuclear-cytoplasmic shuttling in vivo. However, although 14-3-3 binding of HDAC4 has been proposed to be important for its cytoplasmic retention, we find this interaction to be independent of CaMKIV. Rather, the HDAC4.14-3-3 complex exists in the nucleus and is required to confer CaMKIV responsiveness. Our results suggest that the subcellular localization of HDAC4 is regulated by sequential phosphorylation events. The first event is catalyzed by a yet to be identified protein kinase that promotes 14-3-3 binding, and the second event, involving protein kinases such as CaMKIV, leads to efficient nuclear export of the HDAC4.14-3-3 complex.

Expression and localization of p53 and bcl-2 in healing wounds in diabetic and nondiabetic mice.

In a healing wound, inflammatory cells undergo apoptosis immediately beneath the leading edge of migrating epithelium. A potential mediator of this apoptosis pattern is p53, a protein with antiproliferative effects. Another protein, bcl-2, is antagonistic to p53 and prevents apoptosis. The purpose of this study was to determine the expression and location of p53 and bcl-2 mRNA and protein in healing wounds of normal and genetically diabetic mice. At various time points, full-thickness skin wounds from nondiabetic and diabetic mice were evaluated for p53 and bcl-2 by immunohistochemistry and in situ hybridization. Apoptosis patterns were also determined using the TUNEL method. Messenger RNA for p53 and bcl-2 were quantitated by competitive reverse transcriptase-polymerase chain reaction. Protein and mRNA for p53 were expressed in the leading edge of migrating epithelium, with apoptosis patterns closely following those of p53 production. p53 mRNA levels decreased soon after wounding, but after a few days, levels increased to greater than baseline. bcl-2 was localized to the wound epithelium, but relative amounts tended to oppose levels of p53, i.e, when p53 increased, bcl-2 decreased and vice versa. Wounds in diabetic animals showed a delayed onset of p53 mRNA expression but had persistently greater levels for longer periods of time. bcl-2 mRNA expression was further delayed in diabetic mice and did not develop to levels as high as p53. Production of both proteins was delayed, consistent with the mRNA expression. Our data show that immediately after wounding, bcl-2 increases and p53 decreases to allow for the cellular proliferation that is required for tissue repair. Over time, bcl-2 levels decrease while p53 levels increase to shut down the inflammatory process and down-regulate the proliferative response. Diabetic animals appear to lose the indirect relationship between p53 and bcl-2. This loss may contribute to the altered apoptosis patterns observed in diabetic healing.

Activation of orphan receptor-mediated transcription by Ca(2+)/calmodulin-dependent protein kinase IV.

Retinoid-related receptor alpha (RORalpha) is an orphan nuclear receptor that constitutively activates transcription from its cognate response element. We show that RORalpha is Ca(2+ )responsive, and a Ca(2+)/calmodulin-independent form of Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) potentiates RORalpha-dependent transcription 20- to 30-fold. Other orphan receptors including RORalpha2, RORgamma and COUP-TFI are also potentiated by CaMKIV. Transcriptional activation by CaMKIV is orphan receptor selective and does not occur with either the thyroid hormone or estrogen receptor. CaMKIV does not phosphorylate RORalpha or its ligand-binding domain (LBD) in vitro, although the LBD is essential for transactivation. Therefore, the RORalpha LBD was used in the mammalian two-hybrid assay to identify a single class of small peptide molecules containing LXXLL motifs that interacted with greater affinity in the presence of CaMKIV. This class of peptides antagonized activation of orphan receptor-mediated transcription by CaMKIV. These studies demonstrate a pivotal role for CaMKIV in the regulation of orphan receptor-mediated transcription.

Intracellular tryptophan pool sizes may account for differences in gamma interferon-mediated inhibition and persistence of chlamydial growth in polarized and nonpolarized cells.

Gamma interferon (IFN-gamma) is an important factor in the modulating inhibition of intracellular chlamydial growth and persistence. In human epithelial cells and macrophages, this inhibition is the result of depletion of the essential amino acid tryptophan via the IFN-gamma-induced enzyme indoleamine 2, 3-dioxygenase. Under these conditions, chlamydiae must successfully compete with the host cell for limited resources in order to maintain viability. We provide evidence to support the hypothesis that the host cell polarization state influences the host-pathogen interplay and outcome of IFN-gamma-mediated inhibition. In polarized cells, intracellular soluble tryptophan pools were larger than those in nonpolarized cells despite only small differences in the initial uptake rate of this amino acid compared to that in nonpolarized cells. Furthermore, in Chlamydia trachomatis-infected cells, the amounts of tryptophan consumed by the organisms were similar for cells grown in either state. We propose that intracellular tryptophan pool sizes can account for differences in IFN-gamma-mediated chlamydial persistence and growth inhibition in polarized and nonpolarized cells. Collectively, these results argue that polarized cell models, which more accurately reflect the conditions in vivo, may be more relevant than conventionally cultured cells in the study of intimate intracellular host-parasite interactions.

Interleukin-6 production in human intestinal epithelial cells increases in association with the heat shock response.

BACKGROUND: In recent studies, IL-1beta stimulated the production of IL-6 in human enterocytes. The heat shock response influences the production of inflammatory mediators in certain cell types. We tested the hypothesis that heat shock regulates IL-1beta-induced IL-6 production in human intestinal epithelial cells. MATERIALS AND METHODS: Cultured Caco-2 cells, a human intestinal epithelial cell line, were exposed to thermal heat shock at 43 degreesC for 1 h and recovered at 37 degreesC for 1 h. Cells were harvested for analysis of heat shock protein-70 (HSP-70) production by Western blotting. In other experiments, IL-1beta (0.5 ng/ml) was added following heat shock and recovery. IL-6 protein was measured in culture medium after 24 h by enzyme-linked immunosorbent assay and IL-6 messenger RNA (mRNA) levels were measured after 4 h by competitive reverse transcriptase polymerase chain reaction. RESULTS: Heat shock resulted in the production of HSP-70 and potentiated IL-1beta-induced IL-6 production. The response to heat shock was associated with increased IL-6 mRNA levels. CONCLUSIONS: The results suggest that IL-1beta-induced IL-6 production in human enterocytes is increased in association with the heat shock response. The biological role of heat shock-potentiated IL-6 production in the enterocyte remains to be determined.

Differential effects of gamma interferon on Chlamydia trachomatis growth in polarized and nonpolarized human epithelial cells in culture.

The effects of gamma interferon (IFN-gamma) on Chlamydia trachomatis growth in polarized epithelial cells were examined. The range of IFN-gamma concentrations causing aberrant chlamydial growth was wider in polarized than in nonpolarized cultures. Results indicate that chlamydial growth modulation in polarized cells readily leads to persistence and better reflects in vivo conditions.

Ca2+/calmodulin-dependent protein kinase IV and calcium signaling.

Ca2+/calmodulin dependent protein kinase IV (CaMKIV) is a multifunctional, serine-threonine protein kinase that is activated in the presence of increased intracellular calcium (Ca2+). CaMKIV is a potent mediator of Ca2+ induced gene expression, primarily through its ability to phosphorylate and activate transcription factors such as CREB. CaMKIV-dependent activation of CREB is a key event in the expression of genes involved in the processes of T-cell activation and neuronal long term potentiation. The focus of this review is to describe the biochemical regulation of CaMKIV and examine how CaMKIV activates transcription in response to calcium in both cell and animal models.

Differential expression and localization of insulin-like growth factors I and II in cutaneous wounds of diabetic and nondiabetic mice.

Insulin-like growth factor (IGF)-I has profound effects on tissue repair. IGF-II is felt to exert its influence predominately during fetal development. The purpose of this study was to localize and quantify the expression of IGF-I and IGF-II mRNA and protein during early wound healing in diabetic and nondiabetic mice. The hypothesis is that IGF-I and IGF-II are up-regulated in the healing wound, but their expression is inhibited in diabetics. Full-thickness cutaneous wounds were made on genetically diabetic (C57BL/ KsJ-db/db) mice and their nondiabetic littermates. At various times after wounding, one-half of each wound was fixed and paraffin embedded for immunohistochemistry and in situ hybridization. The other half was flash-frozen for quantification of IGF mRNA by competitive reverse transcriptase polymerase chain reaction and protein by radioimmunoassay. IGF-I mRNA rose sharply in nondiabetics at day 3. Expression in diabetic wounds was significantly delayed until 14 days after wounding. Even then, diabetic IGF-I mRNA levels were 50% less than those in the nondiabetics at their peak. Although not usually considered active in adult life, IGF-II mRNA expression was augmented after wounding, peaking at 3 days in nondiabetics. As with IGF-I, diabetic wounds exhibited a delay in IGF-II mRNA expression, with maximal levels at 10 days after wounding. Interestingly, peak concentrations of IGF-II mRNA were four times greater in diabetics versus nondiabetics. Trends in IGF-I protein expression followed the patterns of mRNA expression. IGF-I levels in nondiabetics were initially double those in diabetics and peaked at 5 days. Diabetic wound concentrations of IGF-I did not peak until 21 days after wounding, at which time they rose to nondiabetic levels. IGF-I and IGF-II proteins were localized to the advancing epithelial edge, to the epithelial cells of adjacent hair follicles, and to the granulation tissue of the wounds. IGF-I and IGF-II mRNA expression was noted in the epithelial edge and in the hair follicles adjacent to the wound, paralleling protein expression. Both IGF-I and IGF-II are up-regulated in the healing wound. A delay in IGF-I and -II presence is noted in the diabetic wound. The impairment in tissue repair in diabetic animals is at least partially due to a deficiency in the production of the IGFs.

IL-6 production in human intestinal epithelial cells following stimulation with IL-1 beta is associated with activation of the transcription factor NF-kappa B.

Recent studies suggest that interleukin-1 beta (IL-1 beta) stimulates interleukin-6 (IL-6) production in human intestinal epithelial cells, but the intracellular mechanisms of this response are not known. In other reports, the nuclear factor-kappa B (NF-kappa B) regulated IL-6 production in certain cell types. We tested the hypothesis that IL-6 production in the enterocyte is associated with activation of NF-kappa B. Caco-2 cells, a human intestinal epithelial cell line, were grown in tissue culture whereafter they were treated with IL-1 beta (0.5 ng/ml). Cells were preincubated with pyrrolidine dithiocarbamate (PDTC; 10-500 microM), tosyl-lys-chloromethylketone (TLCK; 10-500 microM), or genistein (25-75 microM), all of which are known inhibitors of NF-kappa B. IL-6 levels in the culture media were measured after 24 hr by enzyme-linked immunosorbent assay (ELISA) and IL-6 messenger RNA (mRNA) levels were determined after 4 hr by competitive reverse-transcriptase polymerase chain reaction (RT-PCR). NF-kappa B activity was determined by electrophoretic gel mobility shift assay (EMSA). PDTC, TLCK, and genistein each inhibited IL-1 beta-induced IL-6 production by the Caco-2 cells in a dose-dependent fashion. These responses were also associated with a decrease in IL-6 mRNA levels. There was no NF-kappa B activity in untreated cells, but the addition of IL-1 beta resulted in the activation of NF-kappa B as determined by EMSA. The results suggest that IL-1 beta-induced IL-6 production in the enterocyte is associated with activation of NF-kappa B. The inhibition of IL-6 production by the NF-kappa B inhibitors indicates that the IL-6 production is regulated by NF-kappa B, although further experiments are needed to test that hypothesis.

Biochemical and crystallographic analyses of a portal mutant of the adipocyte lipid-binding protein.

A number of crystallographic studies of the adipocyte lipid-binding protein have established that the fatty acid-binding site is within an internalized water-filled cavity. The same studies have also suggested the existence of a region physically distinct from the fatty acid-binding site which connects the cavity of the protein with the external solvent, hereafter referred to as the portal. In an effort to examine the portal region, we have used site-directed mutagenesis to introduce the mutations V32D/F57H into the murine ALBP cDNA. Mutant protein has been isolated, crystallized, and its stability and binding properties studied by biochemical methods. As assessed by guanidine-HCl denaturation, the mutant form exhibited a slight overall destabilization relative to the wild-type protein under both acid and alkaline conditions. Accessibility to the cavity in both the mutant and wild-type proteins was observed by stopped-flow analysis of the modification of a cavity residue, Cys117, by the sulfhydryl reactive agent 5, 5'-dithiobis(2-nitrobenzoic acid) at pH 8.5. Cys117 of V32D/F57H ALBP was modified 7-fold faster than the wild-type protein. The ligand binding properties of both the V32D/F57H mutant and wild-type proteins were analyzed using a fluorescent probe at pH 6.0 and 8.0. The apparent dissociation constants for 1-anilinonaphthalene-8-sulfonic acid were approximately 9-10-fold greater than the wild-type protein, independent of pH. In addition, there is a 6-fold increase in the Kd for oleic acid for the portal mutant relative to the wild-type at pH 8.0. To study the effect of pH on the double mutant, it was crystallized and analyzed in two distinct space groups at pH 4.5 and 6.4. While in general the differences in the overall main chain conformations are negligible, changes were observed in the crystallographic structures near the site of the mutations. At both pH values, the mutant side chains are positioned somewhat differently than in wild-type protein. To ensure that the mutations had not altered ionic conditions near the binding site, the crystallographic coordinates were used to monitor the electrostatic potentials from the head group site to the positions near the portal region. The differences in the electrostatic potentials were small in all regions, and did not explain the differences in ligand affinity. We present these results within the context of fatty acid binding and suggest lipid association is more complex than that described within a single equilibrium event.

Regulation and properties of the rat Ca2+/calmodulin-dependent protein kinase IV gene and its protein products.

Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric multifunctional enzyme that is expressed only in subanatomical portions of the brain, T lymphocytes, and postmeiotic male germ cells. It is present in the nucleus of the cells in which it is expressed and can phosphorylate and activate the cyclic AMP response element binding proteins CREB and CREM tau in a manner analogous to protein kinase A. In the absence of Ca2+/calmodulin, CaMKIV is inactive. Activation requires three events: 1) binding of Ca2+/calmodulin; 2) phosphorylation of a single threonine residue present in the activation loop by a separate protein kinase that is also Ca2+/calmodulin-dependent; and 3) autophosphorylation of serine residues present in the extreme N-terminus that is required to relieve a novel form of autoinhibition. The gene for rat CaMKIV has been cloned and found to span 42 kb of DNA. The gene encodes three proteins: namely, the alpha and beta forms of CaMKIV that differ only in that the beta form contains a 28 amino acid N-terminal extension as well as calspermin. Calspermin is the C-terminal 169 amino acids of CaMKIV that binds Ca2+/calmodulin and is expressed only in postmeiotic male germ cells. The promoter for calspermin resides in the penultimate intron of the CaMKIV gene and is regulated by two CREs. This promoter is sufficient to faithfully target expression of a reporter gene to the postmeiotic male germ cells of transgenic mice. Transgene expression can be induced in cells from the transgenic mice that do not normally express it by transfection of CREM tau and CaMKIV. These data suggest that rearrangement of chromatin during meiosis together with the expression of CREM tau at high levels are sufficient to control expression of the calspermin promoter during spermatogenesis. On the other hand, the developmental expression of CaMKIV in brain and thymus appears to be controlled by thyroid hormone mediated via the thyroid hormone receptor alpha. In T lymphocytes, CaMKIV will phosphorylate CREB in response to signals that result in T cell activation. Transgenic mice that express a kinase minus mutant of CaMKIV specifically in thymic T cells show a marked reduction of total thymic cellularity. The remaining T cells undergo a much greater than normal rate of spontaneous apoptosis when placed in culture. These cells fail to generate the signals to phosphorylate CREB and produce significantly less of the cytokine Interleukin-2 (IL-2) in response to agents that either increase intracellular Ca2+ and/or activate protein kinase C. Collectively, the data suggest that CaMKIV may be involved both in preventing apoptosis during T cell development and also in the early cascade of events that is required to activate the mature T cells in response to a mitogenic stimulus.

Expression, purification, and ligand-binding analysis of recombinant keratinocyte lipid-binding protein (MAL-1), an intracellular lipid-binding found overexpressed in neoplastic skin cells.

The keratinocyte lipid-binding protein (KLBP) has been identified on the basis of nucleotide sequence analysis of its cloned cDNA as a new member of the intracellular lipid-binding protein (iLBP) multigene family. To characterize KLBP and determine its ligand-binding properties, its cDNA was subcloned into Escherichia coli, and the protein was overexpressed and purified to homogeneity by a combination of acid extraction, gel permeation, and ion-exchange chromatographies. Purified KLBP exhibited high-affinity binding of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulfonate (1,8-ANS), displaying an apparent dissociation constant of 390 +/- 90 nM (n = 0.74 +/- 0.2). Using an assay based upon displacement of the bound fluorophore, KLBP was found to bind long chain fatty acids most avidly; oleic acid (18:1) bound with an apparent Kd of 248 +/- 12 nM, and arachidonic acid (20:4) exhibited a dissociation constant of 318 +/- 14 nM. As the length of the fatty acid decreased, the binding affinity was reduced; myristic acid (14:0) bound with a K(d) of 1409 +/- 423 nM, but medium-chain (decanoic acid, 10:0) and short-chain (octanoic acid, 8:0) lipids were not bound at all. The protein did not bind prostaglandin E2 with any measurable affinity but did associate with eicosanoids such as 5-hydroperoxyeicosatetraenoic acid (5-HPETE; K(d) of 848 +/- 211 nM) and 15-HPETE (Kd of 463 +/- 243 nM) and to a lesser extent their hydroxy derivatives, 5-HETE and 15-HETE (Kd of 1560 +/- 115 nM and greater than 4 microM, respectively). all-trans-Retinoic acid was a weak ligand for KLBP, binding with a Kd of 3600 nM, and all-trans-retinol did not displace 1,8-ANS. Molecular modeling of the KLBP sequence upon the X-ray crystal structures of several iLBP's suggested that the side chains of one or more cysteine residues may reside within the putative ligand-binding cavity. Consistent with this, sulfhydryl titration of purified KLBP with 5,5'-dithiobis(2-nitrobenzoic acid) at pH 8.0 in the presence and absence of oleic acid revealed that at least one residue was protected from modification by the fatty acid. These results describe the first purification and characterization of the ligand-binding properties of KLBP and indicate that the protein is a fatty acid binding protein with a tertiary structure likely to be similar to other members of the iLBP multigene family.

A simple assay for intracellular lipid-binding proteins using displacement of 1-anilinonaphthalene 8-sulfonic acid.

The fluorescent probe 1-anilinonapthalene 8-sulfonic acid (1,8-ANS) has been used to characterize a general assay for members of the intracellular lipid-binding protein (iLBP) multigene family. The adipocyte lipid-binding protein (ALBP), the keratinocyte lipid-binding protein (KLBP), the cellular retinol-binding protein (CRBP), and the cellular retinoic acid-binding protein I (CRABPI) have been characterized as to their ligand binding activities using 1,8-ANS. ALBP and KLBP exhibited the highest affinity probe binding with apparent dissociation constants (Kd) of 410 and 530 nM, respectively, while CRBP and CRABPI bound 1,8-ANS with apparent dissociation constants of 7.7 and 25 microM, respectively. In order to quantitate the fatty acid and retinoid binding specificity and affinity of ALBP, KLBP, and CRBP, a competition assay was developed to monitor the ability of various lipid molecules to displace bound 1,8-ANS from the binding cavity. Oleic acid and arachidonic acid displaced bound 1,8-ANS from ALBP, both with apparent inhibitor constants (Ki) of 134 nM, while all-trans-retinoic acid exhibited a sevenfold lower Ki (870 nM). The short chain fatty acid octanoic acid and all-trans-retinol did not displace the fluorophore from ALBP to any measurable extent. In comparison, the displacement assay revealed that KLBP bound oleic acid and arachidonic acid with high affinity (Ki = 420 and 400 nM, respectively) but bound all-trans-retinoic acid with a markedly reduced affinity (Ki = 3.6 microM). Like that for ALBP, neither octanoic acid nor all-trans-retinol were bound by KLBP. Displacement of 1,8-ANS from CRBP by all-trans-retinal and all-trans-retinoic acid yielded Ki values of 1.7 and 5.3 microM, respectively. These results indicate the utility of the assay for characterizing the ligand binding characteristics of members of the iLBP family and suggests that this technique may be used to characterize the ligand binding properties of other hydrophobic ligand binding proteins.

Modulation of ligand binding affinity of the adipocyte lipid-binding protein by selective mutation. Analysis in vitro and in situ.

The crystal structure of the adipocyte lipid-binding protein (ALBP) with coordinated fatty acid shows the hydrophobic ligand bound within a water-filled central cavity with its carboxyl group engaged in a hydrogen bonding network involving, at least in part, the functional groups of residues R126 and Y128. We produced mutant forms of ALBP which altered these amino acids, expressed these in Escherichia coli as glutathione S-transferase (GST) fusion proteins, and examined their ligand-binding properties using the fluorescent fatty acids cis-parinaric acid (c-PA) and 12-(9-anthroyloxy)-oleate (12-AO). The wild-type and all mutated forms of GST-ALBP displayed similar binding affinities for 12-AO, with Kd,app values ranging from 0.5 to 2.4 microM. The binding affinity of ALBP forms R126Q and Y128W for c-PA were reduced about 30-50-fold in comparison to GST-ALBP, while that for the double mutation R126L + Y128F was below the limits of detection. To determine if the hydrogen bonding system functioned in situ, Chinese hamster ovary (CHO) cell transfectants expressing wild-type ALBP demonstrated a moderate (1.5-2-fold) increase in the total rate of [3H]oleate uptake and trafficking into the esterified lipid pools over that of untransfected cells, while the rate of [3H]oleate uptake of the transfected CHOs expressing the R126L + Y128F mutation was identical to that of the control CHOs. In summary, these results suggest that the primary factor contributing to binding affinity of ALBP for fatty acids such as c-PA or oleic acid both in vitro and in situ is the hydrogen bonding network involving at least R126, Y128, and the lipid carboxyl group. However, a ligand with sufficiently large hydrophobic character such as 12-AO can bind in the absence of a functional carboxylate hydrogen bonding network, presumably due to stabilizing entropic interactions with other cavity atoms.